Determination of 2-chloro-2'-deoxyadenosine nucleotides in leukemic cells by ion-pair high-performance liquid...

A specific isocratic ion-pair HPLC method for the quantitation of mono-, di- and triphosphates of 2-chloro-2'-deoxyadenosine (CdA) in leukemic cells from patients is described. The method is based on an extraction of nucleotides from cells with a solution of perchloric acid containing triethylammonium phosphate followed by an isocratic separation on an Ultrasphere ODS column (250 x 4.6 mm, 5 microns) with a mixture of 89% triethylammonium phosphate buffer (0.08 M, pH 6.1) and 11% methanol as the eluent. UV absorbance at 265 nm was used. The limit of detection was 65 nM. Standard curves for the CdA triphosphate (CdATP) were linear within the concentration range of 200 nM to 12 microM. The mean overall recovery of CdATP was 90% within a concentration range of standard curves. The within-day and day-to-day coefficients of variation at concentrations of 1.44 microM and 6.25 microM CdATP were < 10%. The applicability of the method was demonstrated by in vitro studies of the accumulation of CdA mono-, di- and triphosphates in CCRF-CEM cells and by determination of the cellular pharmacokinetics of CdA nucleotides in leukemic cells from a patient treated with CdA.